Hypoxia promotes osteogenesis by facilitating acetyl‐CoA‐mediated mitochondrial–nuclear communication

Abstract Bone‐derived mesenchymal stem cells (MSCs) reside in a hypoxic niche that maintains their differentiation potential. While hypoxia (low oxygen concentration) was reported to critically support stem cell function and osteogenesis, the molecular events triggering changes in stem cell fate decisions in response to normoxia (high oxygen concentration) remain elusive. Here, we study the impact of normoxia on mitochondrial–nuclear communication during stem cell differentiation. We show that normoxia‐cultured murine MSCs undergo profound transcriptional alterations which cause irreversible osteogenesis defects. Mechanistically, high oxygen promotes chromatin compaction and histone hypo‐acetylation, particularly on promoters and enhancers of osteogenic genes. Although normoxia induces metabolic rewiring resulting in elevated acetyl‐CoA levels, histone hypo‐acetylation occurs due to the trapping of acetyl‐CoA inside mitochondria owing to decreased citrate carrier (CiC) activity. Restoring the cytosolic acetyl‐CoA pool remodels the chromatin landscape and rescues the osteogenic defects. Collectively, our results demonstrate that the metabolism–chromatin–osteogenesis axis is perturbed upon exposure to high oxygen levels and identifies CiC as a novel, oxygen‐sensitive regulator of the MSC function.

. Metabolic profiling of hypoxic and normoxic MSCs.
A, B Glucose consumption (A) and lactate production (B) were measured in the media of hypoxia-and normoxia-cultured cells using the Vi-Cell MetaFLEX instrument. n = 3 biologically independent experiments. C, D Basal (C) and maximal (D) ECAR in hypoxia-and normoxia-cultured MSCs. n = 3 biologically independent experiments. E qRT-PCR analysis of glycolytic genes in hypoxic and normoxic cells. b-actin was used as an internal control for normalization. n = 3 biologically independent experiments. F, G Basal (F) and maximal (G) OCR in hypoxia-and normoxia-cultured MSCs. n = 3 biologically independent experiments. H MFI of hypoxia-and normoxia-cultured cells after staining with the MitoTracker Deep Red FM dye. n = 4 biologically independent experiments.
Data information: Results are shown as mean AE SEM and statistical significance was determined using a two-sided unpaired t-test. Source data are available online for this figure.

EV3
The EMBO Journal e111239 | 2022 Ó 2022 The Authors Nile Red (a.u.) Figure EV3. Impaired lipogenesis of normoxiacultured MSCs is not due to lower CiC levels.
A, B Representative images (A) and quantification of lipid droplets after observing cells under the electron microscope (left) and after staining lipids with Nile Red (right-B). Scale bars, 2 lm for electron microscopy images and 50 lm for confocal images. n = 4 biologically independent experiments and merged results are shown in (B). Results are shown as mean AE SEM and statistical significance was determined using a two-sided unpaired t-test. C Representative images of hypoxia-and normoxia-cultured cells after immunostaining against SREBP1. Scale bar, 50 lm. D Representative immunoblots against FASN and ACC1 in hypoxia-and normoxia-cultured cells.
b-actin was used as a loading control. n = 3 biologically independent experiments.
Source data are available online for this figure. Ó

EV5
The  Figure EV5. Impaired CiC function in normoxic MSCs.
A qRT-PCR analysis of Slc25a1, which encodes citrate carrier. b-actin was used as an internal control for normalization. n = 3 biologically independent experiments. Results are shown as mean AE SEM and statistical significance was determined using a two-sided unpaired t-test. B Representative images of hypoxia-and normoxia-cultured cells after immunostaining against CiC. Scale bar, 50 lm. C Representative images after staining hypoxic and normoxic cells using an anti-FLAG antibody. Cells were transfected with either a vector plasmid or a FLAG-CiCexpressing plasmid. TOMM20 was used as a counterstain for mitochondria to confirm proper localization of the exogenously expressed CiC-FLAG protein, as shown in the magnified inset. Transfection and all downstream experiments were done for n = 2 biologically independent experiments. Scale bar; 50 lm. D-F Representative images after staining cells used in (C) against acetyl-lysine and TOMM20 (D), assessment of the localization, as described above (E), and quantification of nuclear acetyl-lysine signal MFI (F). Nuclei were stained with DAPI. Quantification of nuclear acetyl-lysine MFI from n = 43 hypoxic, n = 63 normoxic and n = 38 normoxic_CiC OE individual cells from a representative experiment of two biologically independent experiments is shown in (F). Results are shown as mean AE SEM and statistical significance was determined with ordinary one-way ANOVA, using the Holm-Sidak's multiple-comparisons test in Panels (E) and (F). The distribution of data points in (F) is shown as a violin plot, where the mean is indicated by a solid line and the quartiles are indicated with dashed lines. In magnified insets, the intensity of the acetyl-lysine signal was adjusted similarly to all samples, for visualization purposes. Scale bar, 25 lm.
Source data are available online for this figure.